1
5 Cell characterization 3 T cell transduction 4 T cell expansion CliniMACS Prodigy® • Fully automated and closed cell production from sample to formulation • Integrated enrichment or depletion by cell surface markers • Instant up- and out-scaling capability with easy parameterization 1 T cell selection The long-term expression of CARs relies on a stable genomic insertion. Typically, two types of viral vectors are used: • Gamma-retroviral vectors (RV) • Lentiviral vectors (LV) MACS GMP Vectofusin-1® can be used with spinoculation to enhance retroviral transduction. Both transduction processes are completely compatible with the CliniMACS Prodigy for efficient transduction of activated T cells. Figure 3: Transduction efficiency of enriched T cells activated with MACS GMP T Cell TransAct is greater than 40% with retroviral (A) or lentiviral (B) vector.Spinoculation with MACS GMP Vectofusin-1 assists with retroviral transduction efficiency. Lentiviral transduction efficiency is improved by transducing T cells the day after stimulation with MACS GMP T Cell TransAct. Clinical-scale expansion of transduced T cells is essential for a CAR T cell product. Optimal cultivation and expansion of transduced T cells rely on the strong synergy of MACS® GMP T Cell TransAct™, TexMACS™ GMP Medium, and MACS GMP Cytokines. CAR T cells can be expanded in TexMACS GMP Medium supplemented with IL-7 and IL-15 without the need for additional human AB serum or animal derived components. Figure 4: CAR T cells were expanded in serum-free TexMACS GMP Medium or TexMACS GMP Medium supplemented with 3% human AB serum. Cell count (A) and viability (B) were measured up to 13 days and cells were cultured in the presence of IL-7 and IL-15. Cell density and viability were similar between serum-free TexMACS Medium or supplemented with human AB serum. The T cell culture was monitored at different time points and multiple runs. Figure 5: The CliniMACS Prodigy® TCT Process expands CAR T cells and provides a favorable phenotype of the final product. Expanded T cells from healthy donor or patient material show large frequencies of early differentiated T cells, such as central memory T cells. 100 Frequency (%) 80 60 40 20 TN TSCM TCM TEM TEFF TN TSCM TCM TEM TEFF Healthy donor Patient material The quality of the CAR T cell product needs to be carefully monitored during and after expansion. Our broad range of tools for flow cytometry like the MACSQuant® Analyzer 10 and REAfinity™ Recombinant Antibodies allow for a detailed analysis of CAR T cells. Figure 6: The TCT Process is optimized for expansion of CAR T cells in serum-free conditions. T cells were activated with MACS GMP T Cell TransAct and transduced with CAR lentiviral vector. TexMACS GMP Medium supplemented with IL-7 and IL-15 was used to cultivate T cells for 13 days. A significantly greater frequency (A) and number (B) of CAR T cells were detected when expanded following the TCT process in the absence of human AB serum or animal derived components. 2 T cell activation 72 h after activation Healthy donor Patient 24 h after activation Activation of T cells is essential for successful viral transduction. MACS® GMP T Cell TransAct™ is a colloidal polymeric nano- matrix that ensures physio- logical and effective stimulation of T cells while maintaining high cell viability. Key benefits of MACS GMP T Cell TransAct are: • Volumetric dosing • Removal by simple washing • Can be sterile filtered Figure 2: Cluster formation of activated CD4 + and CD8 + T cells from a healthy donor (upper row) and a melanoma patient (bottom row). T cells were activated with MACS GMP T Cell TransAct and cultured in TexMACS™ GMP Medium supplemented with MACS GMP Recombinant Human IL-7 and IL-15. Pictures were taken with the integrated microscope camera of the CliniMACS Prodigy 24 and 72 hours after activation. Untransduced Day 0 Day 1 Day 2 Day 3 Transduction efficiency (%) 20 0 40 80 60 100 Lentiviral transduction B A well-defined enriched population is key for producing CAR T cells and provides: • Specific expansion • Higher reproducibility T cells are automatically labelled and selected with CliniMACS® CD4 and CliniMACS® CD8 Reagents. Figure 1: Automated enrichment of CD4 + and CD8 + T cells from leukapheresis (LP) samples is performed by the CliniMACS Prodigy and results in a single-cell suspension of over 90% purity of T cells – the optimal starting material for transducing T cells. 100 T cells (%) Healthy donor 75 50 25 LP Enrichment 0 Granulocytes B cells Monocytes NK cells NKT cells T cells Serum-free With serum A B Cell count (×10 8 ) Time (days) 80 60 40 20 2 2 4 4 6 6 8 8 10 10 12 12 14 14 Viability (%) Time (days) 100 60 80 40 20 0 0 See how easy CAR T cell production is! Static transduction Spinoculation A Donor B RD 114 Donor A GALV 60 40 20 Retrovirus only Retrovirus only Retrovirus + Vectofusin-1 Retrovirus + Vectofusin-1 0 Transduction efficiency (%) Donor A RD 114 Donor B GALV Treatment of cancer patients with T cells expressing a Chimeric Antigen Receptor (CAR) is one of the most promising adoptive cellular therapy approaches. 1,2 Reproducible production of these genetically modified T cells in high-quality and clinical-grade is a prerequisite for a wide range of applications. With the CliniMACS Prodigy® all complex cell production steps can be processed automatically in a closed system. 3,4,5 This simplifies logistics and potential contamination issues for the sensitive patient material. 1. Anurathapan, U. et al. (2014) Cytotherapy 16: 713–733. 2. Maus, M.V. et al. (2014) Blood 123: 2625–2635. 3. Mock, U. et al. (2016) Cytotherapy 18: 1002–1011. 4. Lock, L. & Mockel-Tenbrinck, N. et al. (2017) Hum. Gene Ther. 28: 914-925. 5. Priesner, C. et al. (2016) Hum. Gene Ther. 27: 860–869. Frequency of CAR T cells (%) 80 60 40 With serum Serum free 20 0 ** A With serum Serum free Number of CAR T cells 5×10⁹ ** 4×10⁹ 3×10⁹ 2×10⁹ 1×10⁹ 0 B 130-117-889 Background image Copyright © Eye of Science Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS® GMP Products are for research use and ex vivo cell culture processing only, and are not intended for human in vivo applications. For regulatory status in the USA, please contact your local representative. MACS GMP Products are manufactured and tested under a quality system certified to ISO 13485 and are in compliance with relevant GMP guidelines. They are designed following the recommendations of USP <1043> on ancillary materials. The CliniMACS® System components, including Reagents, Tubing Sets, Instruments, and PBS/EDTA Buffer, are designed, manufactured and tested under a quality system certified to ISO 13485. In the EU, the CliniMACS System components are available as CE-marked medical devices for their respective intended use, unless otherwise stated. The CliniMACS Reagents and Biotin Conjugates are intended for in vitro use only and are not designated for therapeutic use or direct infusion into patients. The CliniMACS Reagents in combination with the CliniMACS System are intended to separate human cells. Miltenyi Biotec as the manufacturer of the CliniMACS System does not give any recommendations regarding the use of separated cells for therapeutic purposes and does not make any claims regarding a clinical benefit. For the manufacturing and use of target cells in humans the national legislation and regulations – e.g. for the EU the Directive 2004/23/EC (“human tissues and cells”), or the Directive 2002/98/EC (“human blood and blood components”) – must be followed. Thus, any clinical application of the target cells is exclusively within the responsibility of the user of a CliniMACS System. In the US all products of the CliniMACS Prodigy Product Line are available for use only under an approved Investigational New Drug (IND) application. CliniMACS MicroBeads are for research use only and not for human therapeutic or diagnostic use. CliniMACS, CliniMACS Prodigy, MACSQuant, REAfinity, TexMACS, TransAct, Vectofusin, MACS, and the MACS logo are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved. miltenyibiotec.com/tct Chimeric Antigen Receptor T cells
